Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC

Harunori Yoshikawa, Mark Larance, Dylan J. Harney, Ramasubramanian Sundaramoorthy, Tony Ly, Thomas Owen-Hughes, Angus I. Lamond (Lead / Corresponding author)

Research output: Contribution to journalArticle

1 Citation (Scopus)
72 Downloads (Pure)

Abstract

We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

Original languageEnglish
Article numbere36530
Pages (from-to)1-26
Number of pages26
JournaleLife
Volume7
Issue number7
DOIs
Publication statusPublished - 10 Aug 2018

Fingerprint

Polyribosomes
Tissue
Size exclusion chromatography
Ribosome Subunits
Liver
Electron microscopy
Cells
Throughput
Amino Acids
Tissue Extracts
Starvation
Proteins
Cell Extracts
Ribosomes
Proteomics
Gel Chromatography
Electron Microscopy
Cell Line
Injections

Keywords

  • Amino Acids/chemistry
  • Animals
  • Cell Line
  • Chromatography, High Pressure Liquid
  • Humans
  • Mice
  • Polyribosomes/chemistry
  • Protein Biosynthesis
  • Proteomics
  • Ribosomes/chemistry

Cite this

@article{7af3b69a0ac5445380643f8d134889d3,
title = "Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC",
abstract = "We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.",
keywords = "Amino Acids/chemistry, Animals, Cell Line, Chromatography, High Pressure Liquid, Humans, Mice, Polyribosomes/chemistry, Protein Biosynthesis, Proteomics, Ribosomes/chemistry",
author = "Harunori Yoshikawa and Mark Larance and Harney, {Dylan J.} and Ramasubramanian Sundaramoorthy and Tony Ly and Thomas Owen-Hughes and Lamond, {Angus I.}",
note = "Wellcome; H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (MSCA); Uehara Memorial Foundation; Naito Foundation; Royal Society of Edinburgh (RSE)",
year = "2018",
month = "8",
day = "10",
doi = "10.7554/eLife.36530",
language = "English",
volume = "7",
pages = "1--26",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications Ltd.",
number = "7",

}

Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC. / Yoshikawa, Harunori; Larance, Mark; Harney, Dylan J.; Sundaramoorthy, Ramasubramanian; Ly, Tony; Owen-Hughes, Thomas; Lamond, Angus I. (Lead / Corresponding author).

In: eLife, Vol. 7, No. 7, e36530, 10.08.2018, p. 1-26.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC

AU - Yoshikawa, Harunori

AU - Larance, Mark

AU - Harney, Dylan J.

AU - Sundaramoorthy, Ramasubramanian

AU - Ly, Tony

AU - Owen-Hughes, Thomas

AU - Lamond, Angus I.

N1 - Wellcome; H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (MSCA); Uehara Memorial Foundation; Naito Foundation; Royal Society of Edinburgh (RSE)

PY - 2018/8/10

Y1 - 2018/8/10

N2 - We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

AB - We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

KW - Amino Acids/chemistry

KW - Animals

KW - Cell Line

KW - Chromatography, High Pressure Liquid

KW - Humans

KW - Mice

KW - Polyribosomes/chemistry

KW - Protein Biosynthesis

KW - Proteomics

KW - Ribosomes/chemistry

U2 - 10.7554/eLife.36530

DO - 10.7554/eLife.36530

M3 - Article

C2 - 30095066

VL - 7

SP - 1

EP - 26

JO - eLife

JF - eLife

SN - 2050-084X

IS - 7

M1 - e36530

ER -